Method for predicting the response of non-small cell lung cancer patients to targeted pharmacotherapy

ABSTRACT

The present invention relates to a method for determining or predicting the response of a patient diagnosed with non small cell lung cancer to targeted pharmacotherapy. The present invention also aims to provide methods and devices for predicting the response of patients diagnosed with non small cell lung cancer to specific medicaments. More specifically, the present invention provides methods which measure kinase activity by studying phosphorylation levels and profiles and inhibitions thereof by drugs in samples of said patients.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage entry under 35 U.S.C. §371 ofPCT International Patent Application No. PCT/EP2010/054772, filed Apr.12, 2010, and claims priority to European Patent Application No.09157823.7 filed Apr. 10, 2009, the contents of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a method for determining or predictingthe response of a patient diagnosed with non small cell lung cancer totargeted pharmacotherapy. The present invention also aims to providemethods and devices for predicting the response of patients diagnosedwith non small cell lung cancer to specific medicaments. Morespecifically, the present invention provides methods which measurekinase activity by studying phosphorylation levels and profiles andinhibitions thereof by drugs in samples of said patients

BACKGROUND OF THE INVENTION

At present lung cancer is considered to be one of the most importantcauses of death, especially in adults at the ages from 50 to 69 yearsold. Long term exposure to smoking is the cause of lung cancer for 90%of the cases. Among male smokers, the lifetime risk of developing lungcancer is about 17%; among female smokers the risk is about 11%. Fornon-smokers, the risk of developing lung cancer is about 1%. The maincauses for lung cancer in non-smokers are genetic factors, radon gas,asbestos, air pollution and passive smoking. There are two main types oflung cancer: non-small cell lung cancer (NSCLC) (in about 80% of thecases) and small cell lung cancer (in about 17% of the cases). NSCLC canfurther be classified according to the growth type and spread of thecancer cells. NSCLC can therefore be classified into squamous cellcarcinoma, large cell carcinoma and adenocarcinoma. Adenocarcinoma ismore frequent in women, Asians and non-smokers. Other less common typesof NSCLC are pleomorphic, carcinoid tumor, salivary gland carcinoma, andunclassified carcinoma.

It is generally known that most types of lung cancer have a poorprognosis. The 5 year survival for small cell lung cancer is less than5%. Numbers are better for NSCLC. When the tumor is detected when it isstill small and has not spread to the lymph nodes (Stage IA), the 5 yearsurvival is 60%. This number drops rapidly with increasing size of thetumor and lymph node involvement. An early detection prior to themetastasis of the tumor is therefore very important, especially since atan early stage the tumor may be removed entirely by resection. Mostnon-small cell lung cancers, about 50%, however are only detected aftermetastasis. In these cases the 5-year survival of NSCLC is only 10 to15%. However, even when NSCLC is detected at an early stage, the 5-yearsurvival rate of the patients is low compared to other types of cancer.Even more, it is known that long-term (>5 years) NSCLC patients do notexperience the same length of life and quality of life as theirage-matched peers or other cancer survivors.

When NSCLC is detected at an early stage (IA to IIIA), the tumor isresected. The resection if followed by chemotherapy for larger tumorsand in case the tumor has spread to the lymph nodes (stages II andIIIA). Patients in stage I receive no further treatment. Although thesepatients have a good prognosis based on tumor staging, a largepercentage of patients develop metastases within several months oryears. The consequence is a short survival time after resection. Forthis group of patients, follow up targeted pharmacotherapy treatmentwould be beneficial. Indeed, 30% to 40% of patients treated withfirst-line therapy will subsequently be candidates for second-linetreatment. The United States Food and Drug Administration approvedsecond-line treatments with docetaxel, pemetrexed, and an example of atargeted pharmacotherapy, erlotinib (an Epidermal Growth Factor Receptor(EGFR) inhibitor). Gefitinib, another targeted pharmacotherapy, an EGFRtyrosine kinase inhibitor, currently has only limited clinical use. Thebenefit in overall survival difference as shown in clinical trials was7.5 months for docetaxal versus 4.6 months for control, 8.3 months forpemetrexed versus 7.9 months for control and 6.7 months for erlotinibversus 4.7 months for control.

Clinical trial studies have clearly shown individual patient benefitingfrom erlotinib (commercial name Tarceva) treatment if patients can beselected based on certain response prediction markers. The use of aerlotinib as a neoadjuvant therapy are being evaluated in clinicaltrials in stage I-III NSCLC patients prior to undergoing definitivetreatment with surgery and/or radiation

At present there are however no clinical or analytical tools areavailable to make the distinction between responders and non-respondersprior to deciding which treatment to administer. A few attempts ofpredictive biomarkers have been described in the literature such as EGFRexpression, (assessed by immunohistochemistry, IHC) and c-K-ras genemutations and EGFR mutations. These methods are however highly variable,not robust and show poor predictivity since these screenings are basedon the detection of a limited number of proteins or genes. A smallvariation in the gene or protein expression will have profound effectson the screening method.

In view of the above, there remains a pressing need for methods thatprovide a fast and accurate prediction of the response of a patientdiagnosed with NSCLC to neoadjuvant and adjuvant targetedpharmacotherapy. These methods would enable to provide informationregarding the efficacy of the preoperative targeted pharmacotherapytreatment, and more specifically provide an early determination of themost suited treatment of the NSCLC patient.

The present invention aims at providing methods and devices forpredicting the response of a patient diagnosed with NSCLC to inductiontargeted pharmacotherapy. The present invention also aims to providemethods and devices for predicting the response of patients diagnosedwith NSCLC to specific medicaments. The method of the present inventiontherefore adds to the existing assays currently used to select therapiesin NSCLC patients.

SUMMARY OF THE INVENTION

The present invention provides methods and devices that enable thedetermination of the response of a patient diagnosed with NSCLC totargeted pharmacotherapy by measuring kinase activity of a NSCLC sample.The present invention further shows how the method and devices can beused to predict the response of patients diagnosed with NSCLC tospecific treatments. The method of the present invention therefore addsto the existing assays currently used to select therapies in NSCLCpatients.

The present invention therefore provides a method for determining orpredicting the response of a patient diagnosed with NSCLC to targetedpharmacotherapy and preferably to a medicament. In a first embodiment ofthe present invention, the method comprises the steps of:

(a) measuring kinase activity of a sample, obtained from the non-smallcell lung tumor from said patient, in the presence and in the absence ofsaid medicament, thereby providing a phosphorylation profile of saidsample in the presence of said medicament and a phosphorylation profileof said sample in the absence of said medicament; and,

(b) determining from said phosphorylation profiles in the presence andin the absence of said medicament the differential phosphorylationlevel, said differential phosphorylation level predicting the responseof said patient to said medicament.

In another embodiment according to the present invention, thephosphorylation profiles comprise the phosphorylation levels of,preferably one or more, phosphorylation sites present in at least any ofthe peptide markers as listed in table 1. Preferably phosphorylationlevels will be studied of phosphorylation sites present in at least 1,2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78 or 79 of the peptide markers listed in Table 1.

More preferably the present invention relates to a method according tothe present invention wherein said medicament is a protein kinaseinhibitor. More preferably said protein kinase inhibitor is erlotinib.

Another embodiment of the present invention regards a method forpredicting the response of a patient diagnosed with non-small cell lungcancer to a medicament, wherein the kinase activity of a sample,obtained from the non-small cell lung tumor from said patient, ismeasured in the presence and in the absence of a protein kinaseinhibitor targeting a target identical to the target of said medicamentand wherein said kinase activity in the presence of said protein kinaseinhibitor is compared to the kinase activity in the absence of saidprotein kinase inhibitor thereby determining the response of saidpatient to said medicament, wherein said kinase activity measurementprovides phosphorylation profiles of said sample in the presence and inthe absence of said protein kinase inhibitor.

The present invention also relates according to another embodiment to anarray for carrying out the method of the present invention, said arraycomprising immobilized proteins, peptides or peptide mimeticscomprising, preferably one or more, phosphorylation sites present in anyof the peptide markers as listed in table 1. More preferably said arraycomprises immobilized proteins, peptides or peptide mimetics comprising,preferably one or more, phosphorylation sites present in at least 1, 2,3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78 or 79 of the peptide markers listed in Table 1.

The present invention further relates in yet another embodiment to amethod for prediction of response to a targeted pharmacotherapy fromnon-small cell lung cancer, comprising the steps of:

(a) measuring the kinase activity of a sample, obtained from thenon-small cell lung tumor from said patient, in the presence and in theabsence of said medicament or a protein kinase inhibitor targeting atarget identical to the target of said medicament, thereby providing thephosphorylation level of phosphorylation sites present in any of thepeptide markers as listed in Table 1; and,

(b) determining from said phosphorylation level in the presence and inthe absence of said medicament or a protein kinase inhibitor targeting atarget identical to the target of said medicament the response to saidmedicament of said patient.

These and further aspects and embodiments are described in the followingsections and in the claims.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides, as depicted in the examples, a graphical representationof the prediction for response to NSCLC pharmacotherapy.

DETAILED DESCRIPTION OF THE INVENTION

Before the present method and devices used in the invention aredescribed, it is to be understood that this invention is not limited toparticular methods, components, or devices described, as such methods,components, and devices may, of course, vary. It is also to beunderstood that the terminology used herein is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein may be used inthe practice or testing of the present invention, the preferred methodsand materials are now described.

In this specification and the appended claims, the singular forms “a”,“an”, and “the” include plural references unless the context clearlydictates otherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps.

The terms “comprising”, “comprises” and “comprised of” also include theterm “consisting of”.

The term “about” as used herein when referring to a measurable valuesuch as a parameter, an amount, a temporal duration, and the like, ismeant to encompass variations of +/−10% or less, preferably +/−5% orless, more preferably +/−1% or less, and still more preferably +/−0.1%or less of and from the specified value, insofar such variations areappropriate to perform in the disclosed invention. It is to beunderstood that the value to which the modifier “about” refers is itselfalso specifically, and preferably, disclosed.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The present invention provides methods and devices that enable thedetermination of the response of a patient diagnosed with NSCLC totargeted pharmacotherapy by measuring kinase activity of a NSCLC samplein the presence and absence of the treatment drug. The present inventionfurther shows how the method and devices can be used to predict theresponse of patients diagnosed with NSCLC to that specific or othermedicaments. The method of the present invention therefore adds to theexisting assays currently used to select therapies in NSCLC patients.Preferably, in one embodiment of the present invention, methods areprovided wherein the kinase activity is protein kinase activity. Forpurposes of the present invention, and as used herein the term “enzymeactivity”, “kinase activity” or “protein kinase activity” refer to theformation of reaction product(s) by a certain amount of enzyme, kinaseor protein kinase acting on a substrate during the course of the assay.

Protein kinase activity is referred to as the activity of proteinkinases. A protein kinase is a generic name for all enzymes thattransfer a phosphate to a protein. About three to four percent of thehuman genome contains transcription information for the formation ofprotein kinases. Currently, there are about 518 known different proteinkinases. However, because three to four percent of the human genome is acode for the formation of protein kinases, there may be many moreseparate kinases in the human body.

A protein kinase is a kinase enzyme that modifies other proteins bycovalently coupling phosphate groups to them. This process or activityis also referred to as phosphorylation. Phosphorylation can therefore beregarded as the process of the addition of a phosphate group to asubstrate. Phosphorylation usually results in a functional change of thesubstrate by changing enzyme activity, cellular location, or associationwith other proteins. Up to 30% of all proteins may be modified by kinaseactivity, and kinases are known to regulate the majority of cellularpathways, especially those involved in signal transduction, thetransmission of signals within the cell. The chemical activity of akinase involves removing a phosphate group from ATP or GTP andcovalently attaching it to amino acids such as serine, threonine,tyrosine, histidine, aspartic acid and/or glutamic acid that have a freehydroxyl group. Most known kinases act on both serine and threonine,others act on tyrosine, and a number act on all serine, threonine andtyrosine. The protein kinase activity monitored with the method of thepresent invention is preferably directed to protein kinases actingtowards serine, threonine and/or tyrosine, preferably acting on bothserine and threonine, on tyrosine or on serine, threonine and tyrosineand more preferably the method of the present invention if preferablydirected to protein kinases acting towards tyrosines.

Protein kinases are distinguished by their ability to phosphorylatesubstrates on discrete sequences. These sequences have been determinedby sequencing the amino acids around the phosphorylation sites and areusually distinct for each protein kinase. The recognition sequence oneach substrate is specific for each kinase catalyst.

Because protein kinases have profound effects on a cell, their activityis highly regulated. Kinases are turned on or off by for instancephosphorylation, by binding of activator proteins or inhibitor proteins,or small molecules, or by controlling their location in the cellrelative to their substrates. Deregulated kinase activity is a frequentcause of disease, particularly cancer, where kinases regulate manyaspects that control cell growth, movement and death. Thereforemonitoring the protein kinase activity in tissues can be of greatimportance and a large amount of information can be obtained whencomparing the kinase activity of different tissue samples.

As described in the present invention, the inventors have surprisinglyfound that the response of a patient diagnosed with NSCLC to targetedpharmacotherapy or neoadjuvant targeted pharmacotherapy can be predictedand/or determined on the basis of the measurement of the kinase activityof a lung tumor sample

The measurement of the kinase activity is performed by contacting anon-small cell lung tumor sample with one or more substrates, preferablyprotein kinase substrates, thereby generating a phosphorylation profile.

Said protein kinase substrates as used herein, are preferably peptides,proteins or peptide mimetics. The protein kinase substrates eachcomprise, preferably one or more, phosphorylation sites that can bephosphorylated by the protein kinases present in the sample. Therefore,exposure of a protein kinase substrate to a sample comprising a proteinkinase results in the phosphorylation of one or more of thephosphorylation sites of the protein kinase substrate. Thisphosphorylation activity can be measured using techniques known in theart. Therefore, during the measurement method the kinase enzymes presentin the sample will phosphorylate, preferably one or more, of thephosphorylation sites on one or more protein kinase substrates. Theinventors have observed essential differences between kinase activity ofNSCLC tumors having a different response to targeted pharmacotherapy.Consequently, the inventors have observed that the kinases present in aNSCLC tumor sample will phosphorylate protein kinase substratesdifferently depending on the response to targeted pharmacotherapy.Phosphorylation signals differ between the samples, resulting inphosphorylation patterns that differ depending on response to targetedpharmacotherapy. The effect has been observed to be even moresignificant when phosphorylation profiles and/or levels in the absenceof a protein kinase inhibitor are compared to measurements in thepresence of a protein kinase inhibitor.

For purposes of the present invention, and as used herein the term“pharmacotherapy”, or “pharmacotherapeutics” or “drug treatment” refersto the use of a pharmaceutical drug, also referred to as medicine ormedicament wherein said pharmacotherapy is intended for use in thediagnosis, cure, mitigation, treatment, or prevention of disease.

The present invention therefore provides a method for determining orpredicting the response of a patient diagnosed with NSCLC to targetedpharmacotherapy and preferably to a medicament. In a first embodiment ofthe present invention, the method comprises the steps of:

(a) measuring kinase activity of a sample, obtained from the non-smallcell lung tumor from said patient, in the presence and in the absence ofsaid medicament, thereby providing a phosphorylation profile of saidsample in the presence of said medicament and a phosphorylation profileof said sample in the absence of said medicament; and,

(b) determining from said phosphorylation profiles in the presence andin the absence of said medicament the differential phosphorylationlevel, said differential phosphorylation level predicting the responseof said patient to said medicament.

It is clear that effects of a medicament can be monitored using thismethod. The medicament affects the degree of inhibition, the potencyand/or the selectivity of the kinases in the sample. More peptideinhibition is caused by the larger effect of the medicament on thekinases in the sample and therefore the drug is less selective. Also anincreased peptide inhibition would lead to a larger amount of normaltissues being affected by the drug, making the drug less tumor tissuespecific.

As referred to in the present application non-small cell lung cancer(NSCLC) regards a specific type of lung cancer. About 8 out of 10 casesof all lung cancers are of the non-small cell type. There are 3sub-types of NSCLC: squamous cell carcinoma, adenocarcinoma andlarge-cell or undifferentiated carcinoma. Other less common types ofNSCLC are pleomorphic, carcinoid tumor, salivary gland carcinoma, andunclassified carcinoma.

As used in the present invention, the term “sample” refers to a sampleobtained from an organism (patient) such as human or from components(e.g. tissue or cells) of such an organism. Said sample is preferablyobtained from a patient diagnosed with NSCLC and needs to be derivedfrom the tumor tissue of said patient. More preferably said sample is aNCLC tumor tissue biopsy, vacuum assisted biopsy, fine needle biopsy ormaterial from a resected tumor. Said sample is thereby referred to as a‘clinical sample’ which is a sample derived from a NSCLC patient.

Said tumor tissue sample is preferably a fresh or a fresh frozen sample.

More preferably, said sample refers to a lysate of a NSCLC tumor tissueobtained through tumor tissue biopsy, fine needle biopsy or materialfrom a resected tumor. Alternatively said sample may be obtained fromspecific NSCLC tumor cell lines and in particular cell lysates thereof.

Alternatively said sample may be derived from a tumor sample that hasbeen cultured in vitro for a limited period of time.

In a preferred embodiment of the present invention said sample is asample that has undergone a preparation step prior to the stepsaccording to the method of the present invention. Preferably saidpreparation step is a step where the protein kinases present in saidsample are released from the tissue by lysis. Additionally the kinasesin the sample may be stabilized, maintained, enriched or isolated, andthe measurement of the kinase activity as performed in step (a) occurson the enriched or isolated protein kinase sample. By first enrichingprotein kinases in the sample or isolating protein kinases from thesample the subsequent measurement of the kinase activity will occur in amore efficient and reliable manner. Also the clarity and intensity ofthe obtained phosphorylation signal will be increased as certaincontaminants are being removed during the enriching or isolating step.

As used in the present invention, the term “phosphorylation profile”refers to a data set representative for the phosphorylation levels of,preferably one or more, phosphorylation sites present on the proteinkinase substrates. When measuring the kinase activity of a sample bycontacting said sample with protein kinase substrates a specificphosphorylation profile is obtained. The phosphorylation profile isgenerated by the phosphorylation of the protein kinase substrates withthe protein kinases present in the sample and it comprises the level ofphosphorylation of the phosphorylation sites present on the proteinkinase substrates used. A phosphorylation profile can thus be generatedwhen using at least one protein kinase substrate in different testconditions such as for example by comparing the phosphorylation of asample on one peptide or protein (protein kinase substrate) in thepresence and absence of a protein kinase inhibitor. More frequentlyphosphorylation profiles of a sample will be measured using severalprotein kinase substrates in the same or sequentially carried outexperiments. Preferably, the present invention determines tyrosinekinase activity levels or profiles.

It should be noted that a person skilled in the art will appreciate thatthe methods of the present invention can use phosphorylation profiles asa basis for determining the predicting the response to a medicament of apatient suffering from non-small cell lung cancer. However, thephosphorylation levels of individual protein kinase substrates can alsobe used as a basis for determining or predicting the response to amedicament of a patient suffering from non-small cell lung cancer.

It should be noted that for the measurement of the protein kinaseactivity, ATP or any other phosphate source needs to be added to thesample when it is contacted with the protein kinase substrates. Thepresence of ATP will lead to a phosphorylation of the protein kinasesubstrates. Alternatively, the phosphorylation of the protein kinasesubstrates can be performed in the absence of exogenous ATP. When no ATPis added during the incubation of the sample with the protein kinasesubstrates, the endogenous ATP, the ATP naturally present in the sample,will act as the primary source of ATP.

The phosphorylation level of each of the protein kinase substrates canbe monitored using any method known in the art. The response of theprotein kinase substrates is determined using a detectable signal, saidsignal resulting from the interaction of the sample with the proteinkinase substrates or by for instance measuring mass differences usingmass spectrometry. In determining the interaction of the sample with theprotein kinase substrates the signal is the result of the interaction ofthe phosphorylated substrates with a molecule capable of binding to thephosphorylated substrates. This binding can be detected by e.g. surfaceplasmon resonance or by the molecule being detectably labelled. For thelatter, the molecule that specifically binds to the substrates ofinterest (e.g. antibody or polynucleotide probe) can be detectablylabelled by virtue of containing an atom (e.g. radionuclide), molecule(e.g. fluorescein), or enzyme or particle or complex that, due to aphysical or chemical property, indicates the presence of the molecule. Amolecule may also be detectably labelled when it is covalently bound toor otherwise associated with a “reporter” molecule (e.g. a biomoleculesuch as an enzyme) that acts on a substrate to produce a detectableatom, molecule or other complex.

Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Labels useful inthe present invention include biotin for staining with labelled avidinor streptavidin conjugate, magnetic beads (e.g. Dynabeads'), fluorescentdyes (e.g. fluorescein, fluorescein-isothiocyanate (FITC), Texas red,rhodamine, green fluorescent protein, enhanced green fluorescent proteinand related proteins with other fluorescence emission wavelengths,lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX[Amersham], SYBR Green I & II [Molecular Probes], and the like),radiolabels (e.g. 3H, 125I, 35S, 14C, or 32P), enzymes (e.g. hydrolases,particularly phosphatases such as alkaline phosphatase, esterases andglycosidases, or oxidoreductases, particularly peroxidases such as horseradish peroxidase, and the like), substrates, cofactors,chemilluminescent groups, chromogenic agents, and colorimetric labelssuch as colloidal gold or coloured glass or plastic (e.g. polystyrene,polypropylene, latex, etc.), protein particles or beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, chemiluminescent and radioactive labels may bedetected using photographic film or scintillation counters, andfluorescent markers may be detected using a photodetector to detectemitted light (e.g. as in fluorescence-activated cell sorting).Enzymatic labels are typically detected by providing the enzyme with asubstrate and detecting a coloured reaction product produced by theaction of the enzyme on the substrate. Colorimetric labels are detectedby simply visualizing the coloured label. Thus, for example, where thelabel is a radioactive label, means for detection include ascintillation counter, photographic film as in autoradiography, orstorage phosphor imaging. Where the label is a fluorescent label, it maybe detected by exciting the fluorochrome with the appropriate wavelengthof light and detecting the resulting fluorescence. The fluorescence maybe detected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Also, simple colorimetriclabels may be detected by observing the colour associated with thelabel. Fluorescence resonance energy transfer has been adapted to detectbinding of unlabeled ligands, which may be useful on arrays.

In a particular embodiment of the present invention the response of theprotein kinase substrates to the sample is determined using detectablylabelled antibodies; more in particular fluorescently labelledantibodies. In those embodiments of the invention where the substratesconsist of protein kinase substrates, the response of the protein kinasesubstrates is determined using fluorescently labelledanti-phosphotyrosine antibodies, fluorescently labelledanti-phosphoserine or fluorescently labelled anti-phosphothreonineantibodies. The use of fluorescently labelled anti-phosphotyrosineantibodies or fluorescently labelled anti-phosphoserine or fluorescentlylabelled anti-phosphothreonine antibodies in the method of the presentinvention, allows real-time or semi real-time determination of theprotein kinase activity and accordingly provides the possibility toexpress the protein kinase activity as the initial velocity of proteinkinase derived from the activity over a certain period of incubation ofthe sample on the protein kinase substrates.

The inventors have found that measuring the kinase activity of thesample in the presence and in the absence of a protein kinase inhibitor,enables an even better differentiation between the prediction of theresponse of a targeted pharmacotherapy of NSCLC patients. Thissurprising effect is due to the differences in protein kinase activitybetween different individual patients and their respective tumors. Thedifference can be reduced by comparing the protein kinase activityprofiles in the presence and absence of a protein kinase inhibitorbetween different patients. This enables a more accurate classificationof the prediction of the response of a targeted pharmacotherapy.

The term “differential phosphorylation level” as used herein thereforerefers to a data set comprising comparison data from the phosphorylationprofiles in the presence and in the absence of a protein kinaseinhibitor. The statistical analysis of the differential phosphorylationlevel can be done using multivariate and/or univariate statisticalmethods known in the art.

The differential phosphorylation levels are obtained by (numerically)comparing the peptide phosphorylation levels or profiles in the presenceand in the absence of the protein kinase inhibitor in the same sample,for instance, but not limited to, providing ratios or differences of theprofiles obtained in the presence and the absence of the protein kinaseinhibitor.

In addition, because the differential phosphorylation level is generatedby comparing the phosphorylation levels or profiles of the same samplein the presence and the absence of the protein kinase inhibitor,preferably during a parallel series of measurements run in the sameinstrument, the differential phosphorylation level is surprisingly foundto be less affected by variation, for example biological variation,experimental variation, compared to single phosphorylation levels orprofiles. This provides a more robust, more sensitive, more reproducibleand more reliable method for determining the prediction ofpharmacotherapy response in NSCLC patients. Moreover, the measurement ofthe kinase activity of said sample preferably occurs by contacting saidsample with at least one protein kinase substrate in the presence and inthe absence of a protein kinase inhibitor. Techniques from the prior artoften require the incubation of the cells or tissues with said compoundspreferably in vivo, during the culturing of the cells or tissues orduring a large time period prior to the actual measurement of the kinaseactivity. The present invention provides that the protein kinaseinhibitor is added directly to the sample and preferably directly to thelysate sample. The protein kinase inhibitors are added to the sampleonly just prior to contacting the sample with the protein kinasesubstrates and performing the kinase activity assay. Consequently, theprotein kinase inhibitors are added in vitro at the time the incubationof the lysate sample with the protein kinase substrates is initiated.The present invention therefore provides an in vitro primary screeningtool which allows the use of a single sample which is split into a firstpart that is used for the incubation of the sample in the absence of aprotein kinase inhibitor while a second part of the sample is used forthe incubation of the sample in the presence of a protein kinaseinhibitor.

In another embodiment according to the present invention, thephosphorylation profiles comprise the phosphorylation levels of,preferably one or more, phosphorylation sites present in at least any ofthe peptide markers as listed in table 1. Preferably phosphorylationlevels will be studied of phosphorylation sites present in at least 1,2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78 or 79 of the peptide markers listed in Table 1.

The term “peptide markers” in the context of the present inventionrefers to the fact that the peptides as listed in Table 1 can bepreferably used according to the methods of the present invention tomeasure the phosphorylation levels of phosphorylation sites of saidmarkers in the presence of protein kinase present in samples. Thephosphorylation levels of the individual phosphorylation sites presentin said markers may be measured and compared in different ways.Therefore the present invention is not limited to the use of peptidesidentical to any of these peptide markers as listed in Table 1 as such.The skilled person may easily on the basis of the peptide markers listedin Table 1 design variant peptides compared to the specific peptides insaid Table and use such variant peptides in a method for measuringphosphorylation levels of phosphorylation sites common to said peptidemarkers as listed in Table 1. These variant peptides may have one ormore (2, 3, 4, 5, 6, 7, etc.) amino acids more or less than the givenpeptides and may also have amino acid substitutions (preferablyconservative amino acid substitutions) as long as these variant peptidesretain at least one or more of the phosphorylation sites of saidoriginal peptides as listed in said table. Further the skilled personmay also easily carry out the methods according to the present inventionby using proteins (full length or N- or C-terminally truncated)comprising the amino acid regions of the “peptide markers” listed inTable 1 as sources for studying the phosphorylation of sites present inthe amino acid regions of the peptides listed in Table 1. Also theskilled person may use peptide mimetics.

The protein kinase substrates as used in the methods described herein,are meant to include peptides, proteins or peptide mimetics comprising,preferably one or more, of the phosphorylation sites of the peptidemarkers of Table 1. Said one or more phosphorylation sites arespecifically phosphorylated by the protein kinases present in the samplethereby providing a phosphorylation profile. More preferably the proteinkinase substrates (peptides, proteins or peptide mimetics) as used inthe method of the present invention comprise, preferably one or more, ofthe phosphorylation sites present in at least two peptide markers aslisted in Table 1. More particularly said protein kinase substratesrepresent the one or more phosphorylation sites present in at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78 and 79 peptide markers as listed in Table 1. In a morepreferred embodiment the protein kinase substrates comprise or consistof, preferably one or more, phosphorylation sites present in all of thepeptide markers listed in Table 1.

A person skilled in the art will appreciate that the phosphorylationsites present in a single peptide marker as listed in Table 1 enableprediction of pharmacotherapy response in NSCLC patients. However, whenthe number of peptide markers as listed in Table 1 increases, so willincrease the specificity, accuracy and sensitivity of the methodaccording to the present invention. When for example only one proteinkinase substrate comprising the phosphorylation sites of a singlepeptide marker as listed in table 1 is used for prediction ofpharmacotherapy response of a NSCLC patient the accuracy of the methodwill be lower, compared to a method where the prediction ofpharmacotherapy response of a NSCLC patient uses multiple protein kinasesubstrates comprising the phosphorylation sites of multiple peptidemarkers as listed in table 1. The highest method accuracy will beobtained when all protein kinase substrates comprising thephosphorylation sites of all peptide markers as listed in table 1 areused.

TABLE 1 list of 79 peptide markers comprising phosphorylation sites usedfor determining the kinase activity, their sequence and SEQ ID NO Thename of the peptide markers refers to the associated proteins and alsorefers to the start and the end position of the amino acid sequence. SEQID NO Name Sequence 1 SRC8_CHICK_492_504 YQAEENTYDEYEN 2 PGFRB_572_584VSSDGHEYIYVDP 3 PLCG1_764_776 IGTAEPDYGALYE 4 MK10_216_228 TSFMMTPYWTRY5 PAXI_111_123 VGEEEHVYSFPNK 6 RASA1_453_465 TVDGKEIYNTIRR 7EPHA1_774_786 LDDFDGTYETQGG 8 FES_706_718 REEADGVYAASGG 9 FER_707_719RQEDGGVYSSSGL 10 K2C6B_53_65 GAGFGSRSLYGLG 11 KSYK_518_530 ALRADENYYKAQT12 NCF1_313_325 QRSRKRLSQDAYR 13 ERBB2_1241_1253 PTAENPEYLGLDV 14EPHA2_765_777 EDDPEATYTTSGG 15 LAT_249_261 EEGAPDYENLQEL 16SRC8_CHICK_476_488 EYEPETVYEVAGA 17 INSR_992_1004 YASSNPEYLSASD 18MET_1227_1239 RDMYDKEYYSVHN 19 NTRK1_489_501 HIIENPQYFSDAC 20 PDPK1_2_14ARTTSQLYDAVPI 21 NTRK2_696_708 GMSRDVYSTDYYR 22 PAXI_24_36 FLSEETPYSYPTG23 PECA1_706_718 KKDTETVYSEVRK 24 PRRX2_202_214 WTASSPYSTVPPY 25MK01_180_192 HTGFLTEYVATRW 26 FGFR3_753_765 TVTSTDEYLDLSA 27EPOR_361_373 SEHAQDTYLVLDK 28 CDK2_8_20 EKIGEGTYGVVYK 29 PGFRB_1014_1028PNEGDNDYIIPLPDP 30 EPHA7_607_619 TYIDPETYEDPNR 31 CD79A_181_193EYEDENLYEGLNL 32 DCX_109_121 GIVYAVSSDRFRS 33 JAK2_563_577VRREVGDYGQLHETE 34 FAK2_572_584 RYIEDEDYYKASV 35 FRK_380_392KVDNEDIYESRHE 36 PDPK1_369_381 DEDCYGNYDNLLS 37 ANXA1_14_26IENEEQEYVQTVK 38 CDK7_157_169 GLAKSFGSPNRAY 39 ENOG_37_49 SGASTGIYEALEL40 PGFRB_768_780 SSNYMAPYDNYVP 41 MK07_211_223 AEHQYFMTEYVAT 42ODBA_340_352 DDSSAYRSVDEVN 43 MBP_198_210 ARTAHYGSLPQKS 44JAK1_1015_1027 AIETDKEYYTVKD 45 ZAP70_485_497 ALGADDSYYTARS 4641_654_666 LDGENIYIRHSNL 47 NPT2A_501_513 AKALGKRTAKYRW 48EGFR_1165_1177 ISLDNPDYQQDFF 49 VGFR2_1046_1058 DFGLARDIYKDPD 50VGFR2_989_1001 EEAPEDLYKDFLT 51 VGFR2_944_956 RFRQGKDYVGAIP 52ART_004_EAIYAAPFAKKKXC EAIYAAPFAKKK 53 TEC_512_524 RYFLDDQYTSSSG 54TNNT1_2_14 SDTEEQEYEEEQP 55 RB_804_816 IYISPLKSPYKIS 56 ERBB4_1277_1289IVAENPEYLSEFS 57 P85A_600_612 NENTEDQYSLVED 58 FGFR2_762_774TLTTNEEYLDLSQ 59 ERBB2_870_882 LDIDETEYHADGG 60 RET_1022_1034TPSDSLIYDDGLS 61 AMPE_5_17 EREGSKRYCIQTK 62 CBL_693_705 EGEEDTEYMTPSS 63FAK1_569_581 RYMEDSTYYKASK 64 PGFRB_709_721 RPPSAELYSNALP 65MK12_178_190 ADSEMTGYWTRW 66 VGFR2_1052_1064 DIYKDPDYVRKGD 67RAF1_332_344 PRGQRDSSYYWEI 68 DYR1A_312_324 CQLGQRIYQYIQS 69ZBT16_621_633 LRTHNGASPYQCT 70 PTN11_539_551 SKRKGHEYTNIKY 71TYRO3_679_691 KIYSGDYYRQGCA 72 PLCG1_1246_1258 EGSFESRYQQPFE 73MBP_263_275 GRASDYKSAHKGF 74 LAT_194_206 MESIDDYVNVPES 75 EFS_246_258GGTDEGIYDVPLL 76 INSR_1348_1360 SLGFKRSYEEHIP 77 PRGR_786_798EQRMKESSFYSLC 78 EPHB1_771_783 DDTSDPTYTSSLG 79 PP2AB_297_309EPHVTRRTPDYFL

It should further be noted that according to a preferred embodiment ofthe present invention the peptide markers as listed in Table 1 can beused as such for carrying out the methods according to the presentinvention. The present invention however also includes the use ofanalogs and combinations of these peptide markers for use in the methodaccording to the present invention. The peptide marker analogs includepeptide markers which show a sequence identity of more than 70%,preferably more than 80% and more preferably more than 90%.

In yet another embodiment, the present invention relates to a methodaccording to the present invention wherein step (b) is replaced by steps(c) and (d) as provided below. The method according to the presentinvention may therefore comprise the steps of:

(a) measuring kinase activity of a sample, obtained from the non-smallcell lung tumor from said patient, in the presence and in the absence ofsaid medicament, thereby providing a phosphorylation profile of saidsample in the presence of said medicament and a phosphorylation profileof said sample in the absence of said medicament;

(c) comparing said phosphorylation profile of said sample in thepresence of said medicament with said phosphorylation profile of saidsample in the absence of said medicament, thereby determining aclassifier parameter; and,

(d) predicting the pharmacotherapeutical response of said patient tosaid medicament on the basis of said classifier parameter.

By establishing a classifier parameter for determining the prediction ofpharmacotherapy response of the NSCLC patient the method of the presentinvention provides a criterion for analysing the results obtained fromthe method of the present invention. This criterion enables a person toprovide a prediction or prognosis on the basis of a single or limitednumber of data. The person providing the prediction or prognosis doesnot have to interpret an entire set of data, but rather bases hisconclusion on the basis of a single or limited number of criteria.

The term “classifier parameter” as used herein represents adiscriminating value which has been determined by establishing thephosphorylation profile in the presence and in the absence of a proteinkinase inhibitor. Said discriminating value identifies the prediction ofresponse of pharmacotherapy of NSCLC patients. The classifier parameterincludes information regarding the phosphorylation level of severalprotein kinase substrates. Classification is a procedure in whichindividual items are placed into groups based on quantitativeinformation on one or more characteristics inherent in the items (e.g.phosphorylation levels or profiles of a sample) and based on a trainingset of previously labelled items (clinical response to apharmacotherapy). The classifier parameter is calculated by applying a“classifier” to the measured phosphorylation levels of a sample. Basedon the classifying parameter a sample is assigned to (or predicted tobelong to) a class (predicting the pharmacotherapy response of saidpatient). The classifier has been previously determined by comparingsamples which are known to belong to the respective relevant classes.For instance the classifier may be a mathematical function that usesinformation regarding the phosphorylation level of several proteinkinase substrates which individual protein kinase substrates can bestatistically weighted based on the measured phosphorylation level of anumber of protein kinase substrates (or values derived from that).Several methods are known in the art for developing a classifierincluding the neural network (Multi-layer Perceptron), support vectormachines, k-nearest neighbours, Gaussian mixture model, naive bayes,decision tree, RBF classifiers, random forest, disciminant analysis,linear discriminant analysis, quadratic discriminant analysis,discriminant analysis—principal component analysis, partial leastsquares discriminant analysis, generalized distance regression andelastic net classification. The classifier parameter determined in thismanner is valid for the same experimental setup in future individualtests.

It is not relevant to give an exact threshold value for the classifierparameter. A relevant threshold value can be obtained by correlating thesensitivity and specificity and the sensitivity/specificity for anythreshold value. A threshold value resulting in a high sensitivityresults in a lower specificity and vice versa. If one wants to increasethe positive predictive value of the test to determine whether NSCLCpatient will respond to targeted pharmacotherapy, then the thresholdvalue of the test can be changed which as a consequence will decreasethe negative predictive value of the test to determine whether NSCLCcancer patient will not respond to targeted pharmacotherapy. If onewants to increase the negative predictive value of the test to determinewhether NSCLC patient will not respond to targeted pharmacotherapy, thenthe threshold value can be changed in the opposite direction which as aconsequence will decrease the positive predictive value of the test todetermine whether NSCLC patient will respond to targeted pharmacotherapy

It is thus up to the diagnostic engineers to determine which level ofpositive predictive value/negative predictivevalue/sensitivity/specificity is desirable and how much loss in positiveor negative predictive value is tolerable. The chosen threshold levelcould be dependent on other diagnostic parameters used in combinationwith the present method by the diagnostic engineers.

In yet another embodiment, the present invention relates to a methodaccording to the present invention wherein said classifier parameterindicates a response to said medicament or to a targeted pharmacotherapyof said patient if said classifier parameter is above a firstpredetermined threshold level, and wherein said classifier parameterindicates non response to said medicament or to a targetedpharmacotherapy of said patient if said classifier parameter is below asecond predetermined threshold level.

According to another embodiment, the present invention relates to themethod of the present invention wherein said differentialphosphorylation level or said classifier parameter indicates a response,no-response or undetermined or intermediate prediction of saidmedicament or the effect of the targeted pharmacotherapy of saidpatient.

As used in the present application the prediction of response totargeted pharmacotherapy of NSCLC patients is generally divided into twotypes of non-responders and responders and additionally someundetermined or intermediate responders. Whereas responders to atargeted pharmacotherapy will survive longer due to the treatment, thenon-responders to a targeted pharmacotherapy will not benefit from thetargeted pharmacotherapy. The method of the present inventionspecifically enables the distinction between responders andnon-responders to a targeted pharmacotherapy.

In another embodiment, the present invention regards the methodaccording to the present invention wherein said peptide markers are atleast two of the peptide markers selected from the group consisting ofthe peptide markers with any of SEQ ID NO 1 to 10. More particularlysaid protein kinase substrates represent the, preferably one or more,phosphorylation sites present in at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 peptide markers with any of SEQ ID NO 1 to 10. In a more preferredembodiment the protein kinase substrates comprise or consist of,preferably one or more, phosphorylation sites present in all of thepeptide markers with any of SEQ ID NO 1 to 10.

The medicament as used in the method of the present invention can be anykind of chemical substance for instance used in the treatment, cure,prevention, or diagnosis of disease or used to otherwise enhancephysical or mental well-being. Specifically said medicament can be akinase inhibitor, and more preferably a protein kinase inhibitor andmost preferably a small molecule protein kinase inhibitor.

More preferably the present invention relates to a method according tothe present invention wherein said medicament is a protein kinaseinhibitor. More preferably said protein kinase inhibitor is erlotinib.

As used herein, the term “protein kinase inhibitor” refers to a type ofenzyme inhibitor which blocks the action of one or more protein kinases,hence they can be subdivided or characterised by peptides or proteinswhose phosphorylation is inhibited. Examples of protein kinaseinhibitors for use in the method of the present invention are Dasatinib(currently used for the treatment of leukaemia); erlotinib (currentlyused for the treatment of non-small cell lung cancer); gefitinib(currently used for the treatment of non-small cell lung cancer);imatinib (currently used for the treatment of gastrointestinal stromaltumors and leukaemia); lapatinib (currently used for the treatment ofbreast cancer); nilotinib (currently used for the treatment ofleukaemia); sorafinib (currently used for the treatment of renal cellcarcinoma and hepatocellular carcinoma; Sunitinib (currently used forthe treatment of renal cell carcinoma); temsirolimus (currently used forthe treatment of renal cell carcinoma); ABT-869; AEE788; Alvocidib;AP23464; AP23846; AP23848; ARRY-142886; ARRY-334543; AT-7519; Axitinib;AZD0530; AZD1152; BIBW-2992; BIRB-796; BMI-1026; BMS-599626; Bosutinib;Brivanib; Canertinib; CCT129202; Cediranib; CEP-7055; CP-547632;CP-724714; Dovitinib; E7080; Enzastaurin; everolimus; FI-700; Gossypol;HKI-272; HMN-176; HMN-214; INNO-406; JNJ-7706621; KRX-0601; LBW242;Lestaurtinib; Midostaurin; MK-0457; MLN8054; MP-470; Neratinib;ON0123380; ON01910; ON-01910; OSI-930; Pazopanib; PD166326; PD173955;PD180970; Pelitinib; PF-2341066; PHA665752; PHA-739358; PX-866; R-547;Seliciclib; Semapimod; Semaxanib; SNS-032; SU011248; SU014813; SU11248;SU11274; SU14813; Tandutinib; Telatinib; TSU-68; UCN-01; Vandetanib;Vatalanib; VE-465; ZM 447439 and protein kinase inhibitors used inresearch including Tyrphostin-1; Tyrphostin-23; Tyrphostin-51;Tyrphostin-63; AG-1007; AG-1112; AG-1433; RG-13022; SU-1498;I-OMe-Tyrphostin; AG-538; Protein Kinase G inhibitor peptide(Arg-Lys-Arg-Ala-Arg-Lys-Glu); Geldanamycin from Streptomyceshygroscopicus; Lavendustin A; and Genistein. More preferably a proteinkinase inhibitor chosen from the group directed against the epidermalgrowth factor receptor including the protein kinase inhibitorsgefitinib, erlotinib, lapatinib, sorafinib and/or sunitinib.

Additionally, the inventors have further found that by adding a secondprotein kinase inhibitor in step (a) of the method of the presentinvention allows further differentiation between the obtainedphosphorylation profiles. When using both a first and a second proteinkinase inhibitor while measuring the kinase activity, four differentphosphorylation profiles can be obtained: a phosphorylation profile inthe absence of any protein kinase inhibitors, a phosphorylation profilein the presence of the first protein kinase inhibitor, a phosphorylationprofile in the presence of the second protein kinase inhibitor and aphosphorylation profile in the presence of the first and the secondprotein kinase inhibitor.

Another embodiment of the present invention relates to a methodaccording to the present invention wherein said kinase substratescarrying phosphorylation sites are located or immobilized on a solidsupport, and preferably a porous solid support. Preferably saidimmobilized kinase substrates carrying phosphorylation sites will beimmobilized proteins, peptides or peptide mimetics. In a preferredembodiment of the present invention peptides are immobilized on a solidsupport.

As used herein “peptide” refers to a short truncated protein generallyconsisting of 2 to 100, preferably 2 to 30, more preferably 5 to 30 andeven more preferably 13 to 18 naturally occurring or synthetic aminoacids which can also be further modified including covalently linkingthe peptide bonds of the alpha carboxyl group of a first amino acid andthe alpha amino group of a second amino acid by eliminating a moleculeof water. The amino acids can be either those naturally occurring aminoacids or chemically synthesized variants of such amino acids or modifiedforms of these amino acids which can be altered from their basicchemical structure by addition of other chemical groups which can befound to be covalently attached to them in naturally occurringcompounds.

As used herein “protein” refers to a polypeptide made of amino acidsarranged in a linear chain and joined together by peptide bonds betweenthe carboxyl and amino groups of adjacent amino acid residues.

As used herein “peptide mimetics” refers to organic compounds which arestructurally similar to peptides and similar to the peptide sequenceslist in Table 1. The peptide mimetics are typically designed fromexisting peptides to alter the molecules characteristics. Improvedcharacteristics can involve, for example improved stability such asresistance to enzymatic degradation, or enhanced biological activity,improved affinity by restricted preferred conformations and ease ofsynthesis. Structural modifications in the peptidomimetic in comparisonto a peptide, can involve backbone modifications as well as side chainmodification.

For measuring the kinase activity of the sample a large variety ofmethods and formats are known in the art. The kinase activity can forexample be measured using ELISA and multiplex ELISA techniques, blottingmethods, mass spectrometry, surface plasmon resonance, capillaryelectrophoresis, bead arrays, macroarrays, microarrays or any othermethod known in the art. Depending on the type of kinase activitymeasurement method the solid support on which the proteins, peptides orpeptide mimetics are fixed may vary. Whereas in ELISA the protein kinasesubstrates are attached to the surface of the microtiterplates, inmicroarrays the protein kinase substrates are immobilized on and/or inthe microarray substrate.

In a preferred embodiment of the present invention the protein kinasesubstrates are immobilized on an array, and preferably a microarray ofprotein kinase substrates wherein the protein kinase substrates areimmobilized onto a solid support or another carrier. The immobilizationcan be either the attachment or adherence of two or more protein kinasesubstrate molecules to the surface of the carrier including attachmentor adherence to the inner surface of said carrier in the case of e.g. aporous or flow-through solid support.

In a preferred embodiment of the present invention, the array of proteinkinase substrates is a flow-through array. The flow-through array asused herein could be made of any carrier material having orientedthrough-going channels as are generally known in the art, such as forexample described in PCT patent publication WO 01/19517. Typically thecarrier is made from a metal oxide, glass, silicon oxide or cellulose.In a particular embodiment the carrier material is made of a metal oxideselected from the group consisting of zinc oxide, zirconium oxide, tinoxide, aluminium oxide, titanium oxide and thallium; in a moreparticular embodiment the metal oxide consists of aluminium oxide.

Accordingly, in a further embodiment of the present invention said arrayis a Pamchip®.

In a further embodiment, the present invention relates to a methodaccording to the present invention wherein said solid support(microarray) comprises any of the peptides as listed in Table 1immobilized thereto.

In a further embodiment, the present invention relates to a methodaccording to the present invention wherein said solid support(microarray) comprises each of the peptide as listed in Table 1immobilized thereto.

Another embodiment of the present invention regards a method forpredicting the response of a patient diagnosed with non-small cell lungcancer to a medicament, wherein the kinase activity of a sample,obtained from the non-small cell lung tumor from said patient, ismeasured in the presence and in the absence of a protein kinaseinhibitor targeting a target identical to the target of said medicamentand wherein said kinase activity in the presence of said protein kinaseinhibitor is compared to the kinase activity in the absence of saidprotein kinase inhibitor thereby determining the response of saidpatient to said medicament, wherein said kinase activity measurementprovides phosphorylation profiles of said sample in the presence and inthe absence of said protein kinase inhibitor.

By using a protein kinase inhibitor targeting a target identical to thetarget of a medicament, the inventors have found that the response ofthe patient to said medicament can be predicted. This method thereforeallows the use of protein kinase inhibitors which have not beenclinically approved as agents predicting the response of a patient to amedicament, if said protein kinase inhibitor and said medicament aretargeted towards the same target.

It should be noted that the observed response of the patient to saidmedicament can either be a positive response, wherein the medicamentwill improve the treatment of said patient, or a negative response,wherein the medicament has a negative or no influence on the treatmentof said patient.

By measuring the kinase activity of a sample, obtained from thenon-small cell lung tumor from said patient, in the presence and in theabsence of a protein kinase inhibitor, the effect of a medicament to theNSCLC can be assessed. This method was found particularly useful in theprediction of response to said medicament, and to enable the distinctionbetween responders and non-responders in the treatment with saidmedicament. The measurement of the kinase activity of said samplepreferably occurs by contacting said sample with at least one proteinkinase substrate in the presence and in the absence of said proteinkinase inhibitor. Techniques from the prior art often require theincubation of the cells or tissues with said protein kinase inhibitorspreferably in vivo, during the culturing of the cells or tissues orduring a large time period prior to the actual measurement of the kinaseactivity. The present invention provides that the protein kinaseinhibitor is added directly to the sample and preferably directly to thelysate sample. The protein kinase inhibitor is added to the sample onlyjust prior to contacting the sample with the protein kinase substratesand performing the kinase activity assay. Consequently, the proteinkinase inhibitor is added in vitro at the time the incubation of thelysate sample with the protein kinase substrates is initiated. Thepresent invention therefore provides an in vitro primary screening toolwhich allows the use of a single sample which is split into a first partthat is used for the incubation of the sample in the absence of aprotein kinase inhibitor while a second part of the sample is used forthe incubation of the sample in the presence of a protein kinaseinhibitor.

The medicament as used in the method of the present invention can be anykind of chemical substance for instance used in the treatment, cure,prevention, or diagnosis of disease or used to otherwise enhancephysical or mental well-being. Specifically said medicament can be akinase inhibitor, and more preferably a protein kinase inhibitor andmost preferably a small molecule protein kinase inhibitor.

In another embodiment of the present invention the method for predictingthe response of a patient diagnosed with non-small cell lung cancer to amedicament, uses phosphorylation profiles which comprise thephosphorylation levels of, preferably one or more, phosphorylation sitespresent in any of the peptide markers as listed in table 1. Preferablyalso this method will use two or more of said peptide markers asdescribed above. More preferably this method will use at least 1, 2, 3,4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78 or 79 of the peptide markers listed in Table 1.

Phosphorylation levels can also be measured according to the invention,without the necessity to generate phosphorylation profiles thereof. Alsofor this embodiment, the amount and the type of peptides, proteins orpeptide mimetics to be used is as described above.

The present invention also relates according to another embodiment to anarray for carrying out the method of the present invention, said arraycomprising immobilized proteins, peptides or peptide mimeticscomprising, preferably one or more, phosphorylation sites present in anyof the peptide markers as listed in table 1. More preferably said arraycomprises immobilized proteins, peptides or peptide mimetics comprising,preferably one or more, phosphorylation sites present in at least 1, 2,3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78 or 79 of the peptide markers listed in Table 1.

In a preferred embodiment said proteins, peptides or peptide mimeticsare at least 25% of proteins, peptides or peptide mimetics on saidarray. Said arrays may further comprise one or more immobilizedproteins, peptides or peptide mimetics which are used as calibrationmeans for performing the methods according to the present invention.

More particularly said array comprises immobilized proteins, peptides orpeptide mimetics comprising, preferably one or more, phosphorylationsites as described in detail above representing the peptide markers aslisted in table 1. Additionally said proteins, peptides or peptidemimetics are preferably at least 25%, at least 50%, at least 70%, atleast 80%, at least 90% or 100% of the proteins, peptides or peptidemimetics on said array.

The type of arrays to be used according to this embodiment are known inthe art and are further detailed above.

The present invention also relates in another embodiment to a computerprogram product for use in conjunction with a computer having aprocessor and a memory connected to the processor, said computer programproduct comprising a computer readable storage medium having a computerprogram mechanism encoded thereon, wherein said computer programmechanism may be loaded into the memory of said computer and cause saidcomputer to carry out the method according to the present invention.

The present invention further relates to a computer system comprising aprocessor, and a memory coupled to said processor and encoding one ormore programs, wherein said one or more programs instruct the processorto carry out the methods according to the present invention.

The present invention also relates in another embodiment to a kit forpredicting the response to a medicament or a targeted pharmacotherapy ofpatients suffering from non-small cell lung cancer, comprising at leastone array according to the present invention, and optionally a computerreadable medium having recorded thereon one or more programs forcarrying out the method according to the present invention.

The present invention further relates in yet another embodiment to amethod for prediction of response to a targeted pharmacotherapy fromnon-small cell lung cancer, comprising the steps of:

(a) measuring the kinase activity of a sample, obtained from thenon-small cell lung tumor from said patient, in the presence and in theabsence of said medicament or a protein kinase inhibitor targeting atarget identical to the target of said medicament, thereby providing thephosphorylation level of phosphorylation sites present in any of thepeptide markers as listed in Table 1; and,

(b) determining from said phosphorylation level in the presence and inthe absence of said medicament or a protein kinase inhibitor targeting atarget identical to the target of said medicament the response to saidmedicament of said patient.

Since the present inventors have identified a surprisingly useful set ofpeptide markers to be used in methods for determining the prediction ofresponse to a targeted pharmacotherapy of a patient suffering fromnon-small cell lung cancer, the skilled man may carry out any method asdefined above wherein he measures the kinase activity of any of thepeptide markers of Table 1. Also this method may be carried out usingthe amount and type of peptides, proteins or protein mimetics as definedabove. The formats for carrying out these methods are also as for themethods described above.

The present invention is hereafter exemplified by the illustration ofparticular, non-limiting examples.

EXAMPLES Example 1 Example Showing how Responders and Non-Responders toTargeted Pharmacotherapy can be Differentiated According to aPhosphorylation Inhibition Profile

NSCLC patients were recruited in a clinical trial. The purpose of thisPhase II clinical trial was to determine the efficacy of Erlotinib(Tarceva) based preoperative neoadjuvant targeted pharmacotherapy inNSCLC patients. This preoperative targeted pharmacotherapy is emergingas a new option for treatment of NSCLC. In this clinical trialpreoperative targeted pharmacotherapy intends to achieve tumordown-staging or sizing to allow subsequent radical resection. However,targeted pharmacotherapy results in substantial variation of responseswithin the patient population and may cause short-term and long-termcomplications. Patients were treated with targeted pharmacotherapy usingerlotinib (Tarceva) 7 days per week for a period of 3 weeks. Tarcevatreatment was stopped 3 days before the surgical intervention.

Clinical response to Tarceva was determined using CT and PET scans after3 weeks of treatment. Pathological response was determined by thepathologists during the post-surgical pathological examination.

The inventors have surprisingly found that the response to targetedpharmacotherapy can be prognosed by testing inhibition of kinaseactivity by the treatment drug through studying kinase phosphorylationactivity and inhibition levels and such profiles in resection tissuefrom the tumor taken after pharmacotherapy. 14 patients were included inthe study. The tumor content for each of the samples was higher than70%, based on HE staining. 6 samples were from partial or completeresponding patients, 8 samples were from non-responding (stable diseaseor progressive disease) patients.

12 coupes of 10 μm thickness from tumor tissue were lysed in 310microliter Mammalian Extraction Buffer (M-PER) containing phosphataseand protease inhibitors. After 30 minutes of lysis on ice, andcentrifugation for 15 min at 4° C., the supernatants were aliquotted andfrozen. For each sample 7 μg of protein diluted in the lysis solution,was pipetted into a reaction mixture composed of 1×ABL buffer (10×Ablbuffer (New England Biolabs, cat.nr B6050S −100 mM MgCl2, 10 mM EGTA, 20mM DTT and 0.1% Brij 35 in 500 mM Tris/HCl, pH 7.5), 0.1% Bovine SerumAlbumin, 100 μM ATP, 7.5 μg/ml phosphotyrosine antibody to an end volumeof 40 microliter. Before incubation of the lysate reaction mixtures onthe PamChip substrate array a blocking step was carried out on thesubstrate arrays with 2% bovine serum albumin. After washing 3× with Ablbuffer and loading of the lysate reaction mixtures into substrate arrayscomprising 140 protein protein kinase substrates, including the 86protein kinase peptide substrates as listed in Table 1, incubation wascommenced thereby measuring the kinase activity of the sample. Eachtumor tissue lysates was tested in four technical replicates on thesubstrate arrays without inhibitor and in two technical replicates withtwo concentrations of inhibitor. In total 2 times 96 substrate arrayswere used. During 60 cycles of pumping the lysate reaction mixturethrough the array, peptide phosphorylation was detected by an antibodypresent in the lysate reaction mixture. Real time data were obtained bymeasuring fluorescence of the bound anti-phosphotyrosine antibody aftereach 5 cycles. Images of the array were taken during the incubation ofthe array and after 60 cycles of incubation. After 60 cycles ofincubation and imaging, the antibody mixture was removed and the arraywas washed. Images were collected at different exposure times. Signalsfor each spot on the image were quantified. Image quantification anddata processing was conducted with dedicated PamGene software (Evolveand Bionavigator). Subsequent data analysis was performed using Matlab(release 2007B, MathWorks Inc) in short; —negative signals from eachspot was handled by applying an additive transformation by subtractingthe 1% percentile point of the overall signal distribution from the dataand adding 1 with a minimum of 1; —normalization to mean respondersignal per substrate array of a plate containing 96 substrate arraysusing standard methods; —univariate correlation of kinase activityprofiles of 140 kinase protein peptide substrates with Tarceva treatmentresponse; —1-way ANOVA analysis shows 10 peptides (Table 1 number 1 to10) that give a significant (p<0.05) difference over reponse; —Binarytreatment response class prediction was performed using the subset ofsamples form the responders and non-responders classes on a subset of 79peptides (Table 1 number 1 to 79); —Partial Least Squares DiscriminantAnalysis (PLS-DA) was used a the classifier.

The PLS-DA class prediction results using aleave-one-out-cross-validation are shown in FIG. 1. On the Y-axis theprediction for response to NSCLC pharmacotherapy are shown. The samplesare sorted along the X-axis. Samples with a targeted pharmacotherapyresponder are represented by a black square symbol, non-respondertargeted pharmacotherapy samples, by a circular open symbol. Samples areclassified as responders if the prediction >0 and as non-responders ifthe prediction <0. Only one responder sample was misclassified as anon-responder sample. Thus using the PLS-DA one out or six respondersamples was misclassified and eight out of eight non-responders werecorrectly classified

The highest method accuracy was obtained by using all protein kinasesubstrates comprising the phosphorylation sites of all peptide markersas listed in table 1. A subset of 20 protein kinase substrates with SEQID NO 1, 2, 4, 6, 8, 9, 11, 13, 26, 27, 32, 37, 45, 49, 61, 65, 72, 75,76, 79 was constructed by assessing the error rate obtained with leaveone out cross validation (LOOCV) of PLS-DA class prediction as afunction of the number of included peptides. The included peptides wereselected by training a PLS-DA classifier on the training set of eachiteration of the LOOCV including all peptides, subsequently the npeptides with the highest absolute value of the regression coefficientswere selected and a new classifier was trained based on these peptides,a prediction for the test sample was then obtained using the newclassifier. On completion of the LOOCV the error rate was obtained asthe percentage of samples that were incorrectly predicted. Thisprocedure was repeated with the number of peptides n taking values inthe range 5-79 The minimal error rate that was obtained included 20peptides with SEQ ID NO 1, 2, 4, 6, 8, 9, 11, 13, 26, 27, 32, 37, 45,49, 61, 65, 72, 75, 76, 79. The results indicate that these 20 peptidescan be used to provide the minimal error rate when classifying newsamples.

The invention claimed is:
 1. A method for predicting determining theresponse of a patient diagnosed with non-small cell lung cancer (NSCLC)to a medicament, comprising the steps of: (a) providing a solid supporthaving immobilized thereupon at least the 20 protein kinase substratesconsisting of SEQ ID NO: 1, 2, 4, 6, 8, 9, 11, 13, 26, 27, 32, 37, 45,49, 61, 65, 72, 75, 76 and 79; a lysate sample obtained from the NSCLCtumor from said patient; and a medicament; (b) measuring the kinaseactivity of the lysate sample in the presence and in the absence of saidmedicament by contacting a first part of said lysate sample with said atleast 20 protein kinase substrates in the presence of said medicament,and by contacting a second part of said lysate sample with said at least20 protein kinase substrates in the absence of said medicament, therebyproviding a phosphorylation profile of said lysate sample in contactwith and in the absence of said medicament, respectively; and (c)determining from said phosphorylation profiles in contact with and inthe absence of said medicament the lysate sample differentialphosphorylation level in response to the medicament, said differentialphosphorylation level determining the response of said patient to saidmedicament.
 2. The method according to claim 1, wherein the determiningof the differential phosphorylation level of step (c) comprises:comparing said phosphorylation profile of said lysate sample in contactwith said medicament with said phosphorylation profile of said lysatesample in the absence of said medicament, thereby determining aclassifier parameter, said classifier parameter predicting a response,non-response or undetermined or intermediate prediction of the effect ofsaid medicament on said patient's tumor kinase activity.
 3. The methodaccording to claim 1, wherein said differential phosphorylation levelpredicts a response, non-response or undetermined or intermediateprediction of the effect of said medicament on said patient's tumorkinase activity.
 4. The method according to claim 1, wherein saidmedicament is a protein kinase inhibitor.
 5. The method according toclaim 4, wherein the medicament is erlotinib.
 6. The method according toclaim 1, wherein the solid support is a porous solid support.
 7. Amethod for determining the response of a patient diagnosed withnon-small cell lung cancer (NSCLC) to a medicament, wherein the kinaseactivity of a lysate sample, obtained from the non-small cell lung tumorfrom said patient, is measured in the presence and in the absence of aprotein kinase inhibitor targeting a target identical to the target ofsaid medicament, the method comprising: (a) contacting a first part ofsaid lysate sample with at least the 20 protein kinase substratesconsisting of SEQ ID NO: 1, 2, 4, 6, 8, 9, 11, 13, 26, 27, 32, 37, 45,49, 61, 65, 72, 75, 76 and 79, wherein said substrates are immobilizedon a solid support; (b) measuring said kinase activity of said firstpart of said lysate sample in contact with said protein kinaseinhibitor; and (c) comparing the measured kinase activity to the kinaseactivity of a second part of said lysate sample in the absence of saidprotein kinase inhibitor, thereby determining the kinase activityresponse of said patient to said medicament, wherein said measured andcompared kinase activities provide phosphorylation profiles of saidlysate sample in contact with and in the absence of said protein kinaseinhibitor.
 8. The method according to claim 7, wherein saidphosphorylation profiles comprise the phosphorylation levels ofphosphorylation sites present in said at least 20 protein kinasesubstrates.